Stereoselective assembly of C-oligosaccharides via modular difunctionalization of glycals

C-oligosaccharides are found in natural products and drug molecules. Despite the considerable progress made during the last decades, modular and stereoselective synthesis of C-oligosaccharides continues to be challenging and underdeveloped compared to the synthesis technology of O-oligosaccharides. Herein, we design a distinct strategy for the stereoselective and efficient synthesis of C-oligosaccharides via palladium-catalyzed nondirected C1–H glycosylation/C2-alkenylation, cyanation, and alkynylation of 2-iodoglycals with glycosyl chloride donors while realizing the difunctionalization of 2-iodoglycals. The catalysis approach tolerates various functional groups, including derivatives of marketed drugs and natural products. Notably, the obtained C-oligosaccharides can be further transformed into various C-glycosides while fully conserving the stereochemistry. The results of density functional theory (DFT) calculations support oxidative addition mechanism of alkenyl-norbornyl-palladacycle (ANP) intermediate with α-mannofuranose chloride and the high stereoselectivity of glycosylation is due to steric hindrance.

1.In the mechanistic study section, Gibbs free energies were computed at the PBE0-D3(BJ)/def2-TZVP level.However, due to the presence of many electron-negative oxygen and chlorine atoms, diffusion functions should be incorporated in the basis set to improve the accuracy (e.g., def2-TZVPD).
2. In the mechanistic study section, the authors suggested a concerted oxidative addition to a glycosyl chloride substrate.Other potential pathways were ruled out based on results from the IRC calculation and the relaxed scan of the C-Cl bond.However, the IRC calculation confirms the sufficiency of a specific transition state, and the relaxed scan experiment may lead to a local minimum.Because these experiments do not ensure that a specific transition state is the most stable transition state, other reaction pathways, such as the SN2 and SN1 pathways, cannot be excluded from the present computational study only.Although the SN2 pathway can be ruled out from the observed anomeric stereoretention of glycosyl chlorides, the SN1 pathway, proposed by the authors in their former paper (J.Org. Chem. 2020, 85, 11280.),remains plausible.To address this point, the dissociation energy of the corresponding glycosyl cation from the Pd(IV) species should be computed as previously calculated by the authors (CCS Chem. 2020, 2, 1729).
3. In Figure S1, valency "II" is assigned to the palladium metal in transition states.However, this is not accurate according to the chemical definition of valency, and these notations should be omitted from all transition states.4. On page 1, the 7th line of the main text, there is a mention of "alkenylation19-20".However, since reference 20 primarily discussed alkylation, it would be more accurate to state "alkenylation,19 alkylation,20" 5.In Supplementary Information, there are many errors that require corrections.a.The TMS peak of the 1H NMR spectrum of compound 4w is designated as 0.5 ppm.Consequently, chemical shifts in this compound are shifted by +0.5 ppm.b.The 1H NMR peak list of compound 4ae lacks 3 protons.Specifically, only 75 protons are shown, while it should have a total of 78 protons.c.The molecular formulas for HRMS of compounds 4e, 5a, and 8a lack one nitrogen.For example, the correct formula for compound 4e is C43H51"N"NaO10, not C43H51NaO10.d.Although the authors state that "chemical shifts are recorded in ppm relative to the residual solvent signal (TMS δ = 0 for 1H NMR)," the TMS peaks are absent in some 1H NMR spectra (i.e., compound 2n).Additionally, on page S1, there are two sentences about NMR reference.(i.e., "Chemical shifts … signal (TMS δ = 0 for 1H NMR and CDCl3 δ = 77.0 for 13C NMR)." and "Chemical shifts … with TMS (tetramethylsilane) as the internal reference standard".)Given the absence of TMS peaks in 13C NMR charts, the latter sentence should be deleted.
6.The characterization data provided in the Supplementary Information is not adequate for the structural determination.a.Although the NOESY correlations are essential for confirming the depicted C-glycosidic linkages, only the NOESY spectra of compounds 4j, 4p, and 4s are included.The glycosidic stereochemistries of the other compounds should be determined based on the NOESY correlations or the J-values of proton peaks.b.Optical rotations and melting points should be reported for all chiral compounds and solid compounds.c.Some new compounds lack HRMS data (e.g., compounds 2k, 2l, 2h1, 2m, 2n, 2o, 3i, 3k, 3l).d. 1H NMR spectra should consistently be listed with three significant digits.Compounds 4m, 4v, and 4ae have only two significant digits.

Reviewer #3 (Remarks to the Author):
The authors present a new method to synthesize C-oligosaccharides through Catellani-type three-component coupling involving 2-iodoglycals and glycosyl chlorides.The developed method is remarkable because it stereoselectively provides various C-oligosaccharides in a single operation.Moreover, several C-oligosaccharide conjugate molecules were synthesized using this method.Therefore, this work is worthy to publish, but the manuscript and Supporting Information should be thoroughly revised and resubmitted because scholarly descriptions are inappropriate.Additional comments are provided below.
(1) L24, "thusas": Typo? (2) L38-41, "C-oligosaccharides are pharmacologically relevant compounds ~ , rendering them preferred chemotherapy drugs, such as maitotoxin, ~": Maitotoxin is a poisonous compound rather than a drug.The descriptions on maitotoxin should be removed from the main text and Fig. 2. Ref 29 should also be deleted because it describes the total synthesis of hikizimycin.
(3) L45: Ref 35 is inappropriate because a natural product synthesis rather than a Coligosaccharide synthesis was reported.(4) L48, "C-at two anomeric carbons": This part is unclear.
C-oligosaccharides are found in many bioactivity relevant molecules including natural products and drugs.
However, the efficient access to them has been challenging the existing synthetic methods.To solve this problem, the authors established a novel protocol to prepare C-oligosaccharide derivatives, which is featured by palladium-catalyzed three-component Catellani-type reaction between 2-iodoglycals, glycosyl chlorides, and various terminating reagents.The method exhibited excellent stereoselectivity and enjoyed broad substrate scope.Moreover, the reaction mechanism was also investigated by DFT calculation to explain the excellent stereoselectivity of the reactions.In the Supporting Information part, all new compounds have been thoroughly characterized.Thus, the manuscript could be considered to publish in Nat.Commun.provided that the following problems have been properly solved.
Reply: We thank the reviewer for the positive recommendation.Much appreciated! 1) The authors noticed that acetylated and unprotected glycals were not viable substrates for the established 2-iodoglycal-involved Catellani reactions, a reasonable explanation should be provided in the main text; Reply: Thank you very much for your comment.For unprotected glycals, we speculate that the possible reason is that the ester groups have certain coordination effects on palladium (ACS Catal. 2018, 8, 7781-7786).On the other hand, we detected a large number of by-products of O-glycosylation in the 4u system (CCS Chem.2020, 2, 1821-1829.10.31635/ccschem.020.202000445).We speculate that the possible reason is that the reaction is more inclined towards O-glycosylation rather than the Catellani reaction process.Possible reasons have been added to manuscript.(Manuscript page 7, line 2-3) 2) Some products were not pure enough as evidenced from the corresponding spectra provided in the Supporting Information part, particularly for compounds 4d, 4g, 4h, 4i, 4l, 4m, and 4o, which made me suspect the yields as well as stereoselectivity reported in the manuscript; Reply: We apologize for any confusion caused by the impurity of the target products.Compounds 4d, 4g, 4h, 4i, 4l, 4m, and 4o were carefully tested again and purified.We found a small decrease in yield, but no change in stereoselectivity.(Manuscript page 7, Fig. 3.and Supporting Information NMR Spectroscopic Data).
3) The language should be further improved to make the manuscript more readable.For instances, the sentence in line 15 "Density functional theory ……", "thuas" in line 24, "C-at two anomeric carbons" in line 48, "ligands and bases.ethylcrylate." in line 87; "including ethyl tert-butyl" in line 117, the sentence "In addition to ……" in line 123, and the sentence "High-efficiency diastereoselective ……" in line 140; Reply: Thank you for your professional suggestions.We have further improved the language to make the manuscript more readable.The sentence "monofunctionalization thuas far typically involved Heck." was changed to "monofunctionalization typically involves palladium-catalyzed Heck." (Manuscript page 1, line 6-7).
The sentence "In addition, methods that directly provide access to C-at two anomeric carbons are rare."was changed to "In addition, the methods for assembling C-disaccharides that involve directly connecting the anomeric carbon and anomeric carbon are rare."(Manuscript page 2, line 11-12).
The sentence "Compound 4a′ was obtained as a byproduct via direct Mizoroki-Heck coupling between 2iodoglycal, which can be inhibited by screening ligands and bases.ethylacrylate."was corrected to "Compound 4a′ was obtained as byproduct via direct Mizoroki-Heck coupling, which can be inhibited by screening ligands and NBEs." (Manuscript page 4, line 13-14).
In addation, we carefully checked the entire manuscript and corrected some incorrect expressions highlighted in the manuscript.4) In the reference part, the format should be modified.For most of references, the journal names were provided in abbreviation form, while the journal names were presented in the full form in ref. 38,39,42,45,51,53.Most importantly, the journal name for ref.57 was missing.
Reply: Thanks for your corrections.We have corrected above typos in the references.In addition, we have referred to the correct reference format and carefully revised all reference formats.Please see the references.
Reviewer #2 (Remarks to the Author): The authors described palladium-catalyzed nondirected C(sp 2 )-H glycosylation of 2-iodoglycals based on Catellani-type reaction.The reactions showed high diastereoselectivity and broad substrate scope and readily provided a variety of unique C-disaccharides with branched alkenyl chains.The authors proposed a plausible mechanism by applying DFT calculations.Because the manuscript demonstrates the high versatility of the method to access the unprecedented C-disaccharide structures and suggests an interesting mechanism for the stereoretention of the anomeric position, it potentially interests the readers of Nature Communications.However, the following points need to be addressed before the publication.

Reply:
We thank the reviewer for the positive recommendation.Much appreciated! 1.In the mechanistic study section, Gibbs free energies were computed at the PBE0-D3(BJ)/def2-TZVP level.However, due to the presence of many electron-negative oxygen and chlorine atoms, diffusion functions should be incorporated in the basis set to improve the accuracy (e.g., def2-TZVPD).
Reply: Thanks very much for the comment.The def2-TZVPD basis set was used for the single point energy corrections.In addition, to fully reveal the energy changes in homogeneous reactions, the solvation free energies were considered in our revised manuscript.The detailed explanations could be found in the Computational Details in SI.
2. In the mechanistic study section, the authors suggested a concerted oxidative addition to a glycosyl chloride substrate.Other potential pathways were ruled out based on results from the IRC calculation and the relaxed scan of the C-Cl bond.However, the IRC calculation confirms the sufficiency of a specific transition state, and the relaxed scan experiment may lead to a local minimum.Because these experiments do not ensure that a specific transition state is the most stable transition state, other reaction pathways, such as the SN2 and SN1 pathways, cannot be excluded from the present computational study only.Although the SN2 pathway can be ruled out from the observed anomeric stereoretention of glycosyl chlorides, the SN1 pathway, proposed by the authors in their former paper (J.Org.Chem. 2020, 85, 11280.),remains plausible.To address this point, the dissociation energy of the corresponding glycosylcation from the Pd(IV) species should be computed as previously calculated by the authors (CCS Chem.2020, 2, 1729).
Reply: Thanks very much for your professional comment.We calculated the dissociation energy based on Chen's work (CCS Chem. 2020, 2, 1729).The Pd-C1 bond of the Pd(IV) species (F-α) dissociates to form the oxocarbenium ion with absorbing 42.4 kcal/mol, which hints that the Pd(IV) intermediate is configurationally stable.(see Supporting Information for details).
3. In Figure S1, valency "II" is assigned to the palladium metal in transition states.However, this is not accurate according to the chemical definition of valency, and these notations should be omitted from all transition states.
Reply: Thank you for your professional suggestion.We have removed valency "II".(Supporting information, Figure S1) 4. On page 1, the 7th line of the main text, there is a mention of "alkenylation19-20".However, since reference 20 primarily discussed alkylation, it would be more accurate to state "alkenylation,19 alkylation,

20"
Reply: Thank you for your professional suggestion.We have corrected the citation in manuscript.
5. In Supplementary Information, there are many errors that require corrections.
Reply: Thanks for your corrections and we are sorry for our negligence.We have corrected some typos in supporting information.
a.The TMS peak of the 1 H NMR spectrum of compound 4w is designated as 0.5 ppm.Consequently, chemical shifts in this compound are shifted by +0.5 ppm.
Reply: Thanks for your correction.The TMS peak 1 H NMR spectrum and corresponding data of the compound 4w was corrected in supporting information.
b.The 1 H NMR peak list of compound 4ae lacks 3 protons.Specifically, only 75 protons are shown, while it should have a total of 78 protons.
Reply: Thanks for your correction.The peak 1 H NMR spectrum and corresponding data of the compound 4ae was corrected in supporting information.c.The molecular formulas for HRMS of compounds 4e, 5a, and 8a lack one nitrogen.For example, the correct formula for compound 4e is C43H51"N"NaO10, not C43H51NaO10.
Reply: Thanks for your correction.We have corrected above typos in supporting information.4e: "C43H51NaO10" was corrected to "C43H51NNaO10" 5a: "C40H45NaO9" was corrected to "C40H45NNaO9" 8a: "C56H65NaO13" was corrected to "C56H66NO13" d.Although the authors state that "chemical shifts are recorded in ppm relative to the residual solvent signal (TMS δ = 0 for 1 H NMR)," the TMS peaks are absent in some 1 H NMR spectra (i.e., compound 2n).
Additionally, on page S1, there are two sentences about NMR reference.(i.e., "Chemical shifts … signal (TMS δ = 0 for 1H NMR and CDCl3 δ = 77.0 for 13 C NMR)." and "Chemical shifts … with TMS (tetramethylsilane) as the internal reference standard".)Given the absence of TMS peaks in 13 C NMR charts, the latter sentence should be deleted.
Reply: Thanks for your corrections.We have corrected the TMS peak of 1 H NMR spectra in compound 2n.In addition, we also examined other spectras and corrected the TMS peak of 1 H NMR spectra in compound 2l, 3n.
The sentence "Chemical shifts (ppm) were recorded with TMS (tetramethylsilane) as the internal reference standard."was deleted.
6.The characterization data provided in the Supplementary Information is not adequate for the structural determination.
a.Although the NOESY correlations are essential for confirming the depicted C-glycosidic linkages, only the NOESY spectra of compounds 4j, 4p, and 4s are included.The glycosidic stereochemistries of the other compounds should be determined based on the NOESY correlations or the J-values of proton peaks.
Reply: Thank you for your professional suggestion.We added the NOESY spectra of compounds 4d, 4g, 4i, 4o, 4l, 4r, 11a in the supporting information.For other carbohydrate compounds of the same type, the stereoscopic configuration is determined by the chemical shift and J-values of anomeric H and marked in the characterization data.b.Optical rotations and melting points should be reported for all chiral compounds and solid compounds.
Reply: Thank you for your professional suggestion.The optical rotation data of all products and melting points (4e, 4p, 4x, 4ad, 8a, 10a) were supplemented and added in supporting information.Reply: Thank you for your professional suggestion.We have addressed the above issues in supporting information.

4a, [α]
6.This manuscript and Supplementary Information have many errors and typos.Some of them are listed below.
The sentence "Density functional theory (DFT) calculations studies are supportive of a concerted oxidative addition mechanism alkenyl-norbornadiene-palladacycle (ANP) intermediate with an α-mannofuranose chloride and the high stereoselectivity of glycosylation was due to steric hindrance."was changed to "The results of density functional theory (DFT) calculations support oxidative addition mechanism of alkenylnorbornadiene-palladacycle (ANP) intermediate with α-mannofuranose chloride and the high stereoselectivity of glycosylation was due to steric hindrance."(Manuscript page 1. Abstract, line 11-14).
i. Page 5, Table1, the chemical structure of N4.The ester moiety should be oriented in the same plane as olefins.